Aiolos represses CD4+ T cell cytotoxic programming via reciprocal regulation of TFH transcription factors and IL-2 sensitivity

During intracellular infection, T follicular helper (TFH) and T helper 1 (TH1) cells promote humoral and cell-mediated responses, respectively. Another subset, CD4-cytotoxic T lymphocytes (CD4-CTLs), eliminate infected cells via functions typically associated with CD8+ T cells. The mechanisms underlying differentiation of these populations are incompletely understood. Here, we identify the transcription factor Aiolos as a reciprocal regulator of TFH and CD4-CTL programming. We find that Aiolos deficiency results in downregulation of key TFH transcription factors, and consequently reduced TFH differentiation and antibody production, during influenza virus infection. Conversely, CD4-CTL programming is elevated, including enhanced Eomes and cytolytic molecule expression. We further demonstrate that Aiolos deficiency allows for enhanced IL-2 sensitivity and increased STAT5 association with CD4-CTL gene targets, including Eomes, effector molecules, and IL2Ra. Thus, our collective findings identify Aiolos as a pivotal regulator of CD4-CTL and TFH programming and highlight its potential as a target for manipulating CD4+ T cell responses.


Summary
Read, Jones and colleagues analyze Aiolos as a regulator of Tfh cell and CD4-CTL responses upon murine flu infection and during in vitro polarization. They find that Aiolos is necessary for Tfh cell development and antibody production in vivo, and in its absence, CD4 T cells assume a CTL fate. They further demonstrate that Aiolos positively regulates a Tfhcell gene program, including that of Zfp831, TCF-1 and Bcl6, while repressing the expression of IL2Ra and IL-2/STAT5 signaling necessary for expression of the CTL gene program. They conclude that Aiolos serves as a central mediator of Tfh vs CTL development.

Significance
Previous work by the authors demonstrated that Aiolos (Ikzf3) can regulate the expression of Bcl6 in Tfh-like cells, suggesting it promotes their differentiation (Read, J Immunol 2017). The current paper extends this observation, demonstrating that Aiolos positively regulates Tfh cell formation in vivo and in vitro, while reciprocally regulating that of CD4 CTLs, the development of which has not been dissected as done here. These findings are strengths of the paper, alongside dissection of the gene regulatory pathways necessary for the observed phenotypic changes. That said, greater dissection of the phenotypic effects of T-cell intrinsic Aiolos regulation would strengthen the authors' conclusions. While Aiolos target gene expression is investigated, its direct regulatory relationships also could be explored in more depth.
Major points 1. Aiolos deficiency results in disrupted Tfh cell differentiation and antibody production as assessed in Ikzf3 germline deficient mice. While the in vitro experiments suggest this effect is CD4 T cell intrinsic, this is not the case for the in vivo effect. Rather, it could be confirmed using adoptive transfers, bone marrow chimeras, and/or CRISPR gene targeting with adoptive transfer. Such experiments would also better determine Aiolos's functional role in Tfh cells. While the reduced antibody response in germline deficiency is assumed to be secondary to reduced numbers of Tfh cells, their functon(trafficking, cytokine production, CD40L expression and so forth) is not tested. Numbers of cells in addition to percentages should be shown throughout. Moreover, while flow data suggest Aiolos is necessary for GC-Tfh cell development, this is an indirect assessment, which would be better determined by GC staining, ideally including for Aiolos. While deficiency in the latter leads to reduction in Ab production 8 days following flu challenge, this time point largely analyzes early PC formation, not GC output, with the latter not determined.
Such experiments would not only rule out a non-T cell intrinsic effect but would eliminate the possibility the phenotype observed is secondary to manipulation of Tfr cells, reduced in the absence of Aiolos. To this point, the authors' reasoning that the observed 'expansion' of regulatory cells rules out a Tfh cell extrinsic effect directly contradicts their observation that Tfr cells are reduced in the germline mutants, an effect which of course can lead to reduced Tfh cell numbers and output. The only way to resolve this point is to do an experiment separating the effects of Aiolos in Tfh vs. Tfr cells.

Reviewer #2 (Remarks to the Author):
In this manuscript Read KA and colleagues investigated the role of the transcription factor Aiolos in Tfh cells. In a previous study from the same group (Read KA J Immunol 2017), the authors found that Aiolos expression was increased in antigen-specific Tfh cells after influenza infection, compared to antigen-specific effector T cells. This new study now aims at extending on this finding, by showing that in the absence of Aiolos, Tfh cell differentiation in response to influenza antigens was impaired, leading to reduced antibody response. On the other hand, T cells lacking Aiolos that were cultured under Tfh conditions acquired some features of a cytotoxic-like program, including perforin expression. Aiolos deficiency also resulted in increased CD25 expression. While some observations warrant further investigation (for example, the role of Aiolos in CD4-CTLs, or in T cell responses to viral infections), other findings appear to be more incremental and preliminary, and in the absence of more thorough and global analyses I remain uncertain whether some of the conclusions are indeed adequately supported by experimental evidence. 1) Currently this study is not adequately put into the context of previous relevant work on Aiolos published by other labs. Most notably: -Quintana et al (Nature Immunology 2012) showed that Aiolos controlled the differentiation and function of Th17 cells, and that Aiolos was most highly expressed in this subset. In Figure 2a, how does expression of Aiolos in Tfh cells compare to Th17 cells? This information would provide more general insights on its role in T cell differentiation.
-Kuehn et al (J Exp Med 2021) showed that a mutation in AIOLOS identified in human patients was associated to T and B cell abnormalities (including impaired Tfh cell differentiation), recapitulated in a mouse model. In Figure 1, can the authors rule out that reduced antibody production is not due to an intrinsic B cell defect? -Wang JH (Immunity 1998) showed increased proliferation of Aiolos-deficient T cells in response to TCR activation. Can the authors rule out that some of the differences they observe in the current study are not due differences in T cell activation? 2) Figure 3: since the authors performed ATAC-seq on Tfh cells, they should assess if a more comprehensive analysis (rather than just looking at a few pre-selected genes) provides more information about the regulatory roles of Aiolos in Tfh cells. In general, I don't think that based on a few selected snapshots the authors can draw the conclusion that "chromatin accessibility was negatively impacted by the absence of Aiolos". There might be many more regions where chromatin accessibility is positively impacted. And the slight reduction shown could simply reflect experimental variability. Also, how do these differences compare to differences in accessibility at established Aiolos targets, like Il2?
3) Still in Figure 3, a better control of specificity for the ChIP-PCR assay is represented by the Ikzf3-/-Tfh cells, which should be used in comparison with the other experimental groups.
4) The authors could establish if Aiolos indeed directly regulates the expression of some of the identified factors (Bcl6, Il2ra, etc) by using gain-of-function experiments and luciferase reporter assays.
5) The authors state (page 10) that Aiolos directly regulates CD25 expression. However, the authors only showed that the Il2ra transcript is expressed comparatively at higher levels (~2 times higher) in Ikzf3-/-Tfh cells compared to wild-type. How does this translate into the dynamics of CD25 expression at different time points of T cell activation and with different strengths of stimulation? A small upregulation of CD25 expression may end up being modest and primarily linked to increased T cell activation, rather than differentiation. B cells lacking Aiolos for example exhibited an activated phenotype (Wang JH Immunity 1998). 6) Along the same line, while I appreciate that the authors show increased CD25 expression in vivo (Figure 7), this could also be due to lower thresholds of T cell activation in absence of Aiolos. What about the surface expression of other activation markers (both in vivo and in vitro) that are not regulated by Aiolos? Including also Icos, which is central to Tfh biology.

7)
Please also indicate where all the relevant transcripts (including Il2ra, Il2rb, Icos) are located in the volcano plots in Figure 2 and 5. 8) A more in-depth comparison of the RNA-seq data in Tfh and Th1 cells could provide more information about the role of Aiolos in these cells. What is the extent of overlap of the upand down-regulated genes in Tfh and Th1 conditions in the absence of Aiolos? Some Venn diagrams with indicated common and unique genes would already be informative. If mostly the same genes are dysregulated in these two cell subsets upon Aiolos deletion, then the main phenotype may be linked to general T cell activation rather than differentiation. 9) Figure 6F: was Cx3cr1 expression increased also in any of the RNA-seq datasets? Excel files containing at the very least the significant differentially expressed genes and differentially accessible regions should be made available to be able to assess the consistency and quality of the data. The number of biological replicates used in RNA-seq for Tfh cells is not indicated neither in the methods nor in the figure legends. Please show PCAs of all RNA-seq and ATAC-seq experiments. Legend of Figure 3 mentions two replicates for ATAC-seq, which is insufficient to achieve robust results. 10) Based on the CD25 data presented in this manuscript, and in the absence of other information (that is, expression of other activation markers not regulated by Aiolos, functional effects of IL-2 on cell proliferation and so on), the conclusion that "Aiolos represses IL-2 responsiveness" (page 12) is both premature and preliminary, since no direct evidences are presented. Figure 8: is the increased STAT5 phosphorylation actually due to IL-2 stimulation? Is it significant? Does it occur also in Aiolos-deficient Tfh cells? Does it affect T cell responses to IL-2? 12) If Aiolos indeed suppresses CD25 expression, then its retroviral transduction in wt and Aiolos-deficient cells should reduce (or normalize, respectively) CD25 expression and any downstream functional effects.

11) I am uncertain what conclusions can be drawn from
13) The observation that T cells lacking Aiolos express granzyme and perforin is interesting: do they also acquire killing capacity in vitro?
14) It is unclear from the figure legend if the human data shown in Extended Figure 1 show 3 independent donors. I also believe that the graph should show SD and not SEM and paired, rather than unpaired t-test. The authors may want to double-check their statistics throughout the manuscript.

Reviewer #3 (Remarks to the Author):
In the manuscript by Read et al, the authors explore the role of Aiolos in Tfh/CD4 CTL differentiation. In line with their previous publication (Read et al, J. Immunol 2017) the authors find that Aiolos supports Bcl6 and subsequent Tfh differentiaton, in this study by using Aiolos (Ikzf3) deficient mice. The loss of Tfh cells leads to a subsequent increase in CD4 T cells that have cytotoxic capabilities, including perforin and Granzyme B, as well as IFNg. This is attributed to alterations in the chromatin accessibility and binding of Aiolos to Tfh genes, and in contrast, a repressive effect on CD25 leading to increased STAT5 signaling and downstream genes associated with CTL function. While the data are straightforward and good quality, the findings are not surprising in light of prior work from this group and others (Quintana et al, Nat. Immunol. 2012) that Aiolos plays a key role in T cell differentiation by promoting Bcl6, Tfh and inhibiting the IL-2 pathway. There are also several missed opportunities that would bolster the impact of these findings, as well as key deficiencies in experimental design that limit the impact. 2. The authors show increased Tregs (Ext Fig 1E). In light of the role of Aiolos inhibiting CD25, does Aiolos also control Treg formation?
3. How does Aiolos alter chromatin accessibility? Is there increased H3K4me3 or H3K27Ac? Given the use of an in vitro system and the authors ability to perform ChIP, this is addressable and would improve the impact with mechanism of how Aiolos alters gene transcription.
4. NP specific tetramer staining should be shown by flow 5. When is Aiolos upregulated? At what point in T cell differentiation is it required for expression? Tfh cells are thought to form early, and if Aiolos is upstream of Bcl6, it suggests Aiolos is required early.

If Aiolos is upregulated and not expressed in naïve cells, what drives its expression?
7. For all in vivo experiments, cell numbers should be shown as well as percentages.
8. Analysis of ATACseq data is limited to a few genes. Are there global changes in chromatin accessibility?

AUTHORS' SUMMARY:
We sincerely appreciate the thorough feedback from the Reviewers and have made substantial efforts to address their concerns and suggestions. Major concerns included the CD4 + T cell-intrinsic nature of our findings originally obtained from germline Aiolos KO mice, as well as a desire to see more indepth analyses of our RNA-and ATAC-seq data. We now present a substantially revised manuscript including newly generated data, analyses, and significant revisions to the manuscript text. Specifically: 1. We now include use of an adoptive transfer system to identify CD4 + T cell-intrinsic roles for Aiolos in regulating CD4 + T cell programming events. Importantly, findings using this system support a CD4 + T cell-intrinsic role for Aiolos in repressing CD4-CTL responses during influenza virus infection. 2. We have substantially expanded our ATAC-seq analyses by both acquiring additional samples and performing genome-wide analyses to 1) identify statistically significant alterations in chromatin accessibility between wildtype and Aiolos-deficient TFH-and TH1-polarized cells, and 2) perform motif analyses to evaluate DNA binding motifs enriched at sites of statistically significant increases or decreases in accessibility. These analyses have revealed global alterations in chromatin accessibility in the absence of Aiolos in both TH1-and TFH-polarized cells and have identified regions of significantly decreased or increased accessibility (consistent with transcript analyses) at key TFH (Zfp831, Tox, Cd40lg) and CD4-CTL (Prdm1, Eomes, Ifng, Prf1, Gzmb, Il2ra, I2rb) associated loci, respectively. Further, enriched motifs identified at significantly differentially accessible regions include STAT3, STAT5, Blimp-1, and T-box transcription factors. We make the further observation that the STAT5 motif contains the core IkZF DNA binding motif GGGAA, suggesting that Aiolos binding may play a role in antagonizing the activity of STAT5. This possibility is consistent with STAT5 ChIP data presented in Figure 8. All ATAC-seq data have been deposited in GEO, and Reviewer tokens are available at request to view the data. 3. We have expanded our ChIP qPCR analyses to assess an increased number of regions displaying significant differences in chromatin accessibility (both increased and decreased) in the absence of Aiolos. This has led to the identification of novel Aiolos target genes, as well as sites of increased STAT5 enrichment in the absence of Aiolos. Further, we also now analyze alterations in H3K27Ac enrichment at these same sites, to further describe changes to chromatin structure in the absence of Aiolos at TFH and CD4-CTL associated genes. Importantly, these new findings support our original conclusion that Aiolos is a reciprocal regulator of TFH and CD4-CTL programming. 4. We have substantially altered language to ensure 1) both clarity and accuracy of our findings and methods, and 2) that related prior work and implications of our current findings are accurately represented throughout the text.
Additional details of our revisions our presented in the point-by-point response below. We appreciate that the Reviewer found several strengths in our study, but also acknowledge the concerns raised by the Reviewer. We have now obtained new data that strengthen our original conclusions, notably that adoptively transferred Aiolos-KO cells upregulated CD4-CTL-like features in the lungs of influenza infected recipient mice (Fig. 6, Extended Data Fig. 10A-D). We also provide new ATAC-seq and ChIP data that yield further insights into the mechanisms involved (Figs. 3, 7). We appreciate the Reviewer's feedback and agreed that our findings would be strengthened by inclusion of data defining the CD4 + T cell-intrinsic role for Aiolos in regulating TFH and CD4-CTL programming. To this end, we performed adoptive transfer studies and, in line with a CD4 + T cell-intrinsic role for Aiolos in regulating CD4-CTL responses, found that transferred populations in the lung exhibited increased Eomes expression, as well as augmented production of IFN-g and the cytotoxic molecules granzyme B and perforin upon antigen stimulation ( Fig. 6 and Extended Data Fig. 10B-D).

Aiolos deficiency results in disrupted Tfh cell differentiation and antibody production as assessed in Ikzf3 germline deficient mice. While the in vitro experiments suggest this effect is CD4 T cell intrinsic, this is not
As the Reviewer notes, CD4 + T cell-intrinsic roles for Aiolos in regulating both TFH and CD4-CTL programming are indeed supported by our in vitro findings. With specific regard to TFH programming, these now include expanded analyses of ATAC-and RNA-seq data, which indicate 1) that the TFH gene program is significantly disrupted in the absence of Aiolos, including reduced expression of numerous critical transcriptional regulators (Bcl6, Zfp831, Tcf7, Tox) ( Fig. 2A-B), 2) that there are global alterations to the chromatin landscape in Aiolosdeficient cells, including significant reductions in accessibility at TFH genes (Zfp831, Tox, Cd40lg), and enhanced accessibility at CD4-CTL-associated genes (Fig. 3A), and 3) that Zfp831, which exhibits both loss of accessibility and reduced transcript expression, is a direct target of Aiolos in this setting ( Fig. 3B-D). Together, these data support a role for Aiolos in driving TFH programming in a cell-intrinsic manner.
While we performed analyses of TFH populations following adoptive transfer experiments discussed above, results from these experiments were more complex than initially anticipated: First, our germline knockout system indicated that in addition to disrupted TFH generation, loss of Aiolos resulted in significant defects in regulatory T cell populations, including both TREG and TFR cells (Extended Data Fig. 3D-F)-both of which could contribute to the observed TFH deficiency. These findings are important, as they indicate for the first time that Aiolos may function to regulate TFR populations. As our data both in this manuscript and a prior study indicate that Aiolos is important for the regulation of Bcl-6, which is also expressed by TFR cells, it is interesting to speculate that this may represent a shared regulatory mechanism between TFH and TFR populations. In contrast, during adoptive transfer, both TREG and TFR populations are present at normal levels (data which we now include in Extended Data Fig. 10F-G). This may explain, in part, why we did not observe a defect in TFH populations in the lung-draining lymph node (DLN) in this experimental setting (shown in Extended Data Fig. 10E).
Second, while data in the lungs recapitulated our findings from Aiolos-deficient animals (i.e. that loss of Aiolos results in augmented, Eomes-dependent CD4-CTL responses ( Fig. 6 and Extended Data Fig. 10A-D), we did not observe the same phenotype in adoptively transferred populations in the DLN (where TFH populations were also analyzed; Extended Data Fig. 10H-I). This suggests that either use of TCR-transgenic cells, or increased numbers of antigen-specific cells utilized in adoptive transfer, may contribute to alterations in the DLN TFH and CD4-CTL responses, as has previously been reported (Olson et al. Immunol Cell Biol. 2016).
The above findings have now been compiled and are included in the revised version of the manuscript. Thus, while we acknowledge that we cannot rule out the potential role of B cell alterations (due to disrupted Aiolos) in the germline knockout setting, the above observations suggest that TFH regulation in these settings may be more nuanced. Still, our in vitro findings are strongly supportive of a CD4 cell-intrinsic role for Aiolos in positively regulating the TFH gene program. These considerations are detailed in the newest version of the Discussion.

1.b. While the reduced antibody response in germline deficiency is assumed to be secondary to reduced numbers of Tfh cells, their function (trafficking, cytokine production, CD40L expression and so forth) is not tested.
We appreciate this feedback from the Reviewer. Our updated analyses suggest that loss of Aiolos results both in reduced Cd40lg transcript expression and significantly reduced accessibility at several Cd40lg regulatory regions shown in ( Fig. 2A-B, Fig. 3A, Extended Data Fig. 6A). Further, our in vivo findings indicate that Cxcr5 expression is reduced in Aiolos-deficient CD4 + T cell populations, which could have marked effects on the ability of Aiolos-deficient cells to localize to the follicle and interact with B cells, relative to their WT counterparts (Fig. 1B,C). While experiments to more precisely assess TFH cell trafficking and germinal center responses have been delayed due to technical hurdles, we are currently pursuing these analyses in efforts to more thoroughly understand the role of Aiolos in TFH differentiation and associated functional responses.

1.c. Numbers of cells in addition to percentages should be shown throughout.
We agree with the Reviewer and have now included the numbers of evaluated influenza nucleoprotein (NP)specific (or CD45.2 + donor T cell, or CD45.2recipient) populations in each tissue examined (Figs. 1D, 5E, 6G and Extended Data Fig. 3C, 9C, and 10E-F). Importantly, we do not observe substantial differences in the numbers of WT and Aiolos-deficient antigen-specific cells with the exception of the lungs in our germline Aiolos KO studies (Extended Data Fig. 9C). These data are now included and discussed in the revised manuscript. We appreciate this feedback from the Reviewer and agree with the above points. We have now both expanded our analyses and adjusted language discussing interpretation of the data to more accurately reflect our findings. We have not yet examined later timepoints for GC output due to technical hurdles, but do plan to pursue these studies moving forward. However, we do now present data demonstrating that Bcl-6 protein is significantly reduced in Aiolos-deficient cells at an earlier timepoint during influenza virus infection (6 d.p.i.), hinting at an early TFH programming defect (which we are continuing to explore; Extended Data Fig. 3B). We acknowledge that disruptions to both TFR and TREG populations may impact the observed TFH phenotype (shown in Extended Data Figs. 3D-F) and have modified language in both the Results and Discussion of the revised manuscript to address this.

To verify Aiolos targets, the authors used ATAC-seq combined with ChIP-qPCR analysis of WT and mutant cells, finding enrichment in the indicated regions of Tfh-related genes or Th1-associated genes. While compelling, it is unclear which genes are direct Aiolos targets. Also, what is the binding motif for Ikzf3? It is not clear from the data if the binding motif is found in in the binding site of different Tfh associated genes.
Our combined ATAC-seq and ChIP-qPCR analyses have thus far identified Bcl6, Zfp831, and Il2ra as direct Aiolos gene targets in TFH cells (Figs. 3A-D, Extended Data Figs. 6C-D, 13D). We hypothesize that additional Aiolos targets exist and are critical in regulating both TFH and CD4-CTL gene profiles, which we plan to explore on a genome-wide basis in future studies. With regard to the Ikzf3 binding motif (GGGAA), we find this motif proximal to enrichment sites of Aiolos. Further, we also find that regions of significantly altered chromatin accessibility in the absence of Aiolos are enriched for STAT5 sites which, notably, contain the core IkZF binding motif (Figs. 3F-G, 7G). Thus, our data are suggestive of the exciting possibility that Aiolos may function to compete with and antagonize STAT5 activity. These possibilities are detailed in the substantially revised Discussion. Together, our current findings that key TFH transcriptional regulators and Il2ra are bound by Aiolos represents a significant conceptual advance in our understanding of the role Aiolos in CD4 + T cell programming events, as well as TFH and CD4-CTL biology. Data Fig. 4C). We hypothesize that the activity of Aiolos may be altered in mature effector populations vs. early T cell precursors due to altered partner proteins. For example, our earlier work demonstrates that Aiolos interacts with STAT3 downstream of cytokine signals received during effector differentiation (Read et al. J Immunol. 2017). Thus, interaction with STAT3 may alter the effect of Aiolos on gene programs in effector cells versus developing T cells. An intriguing aspect of our data is that Tcf7 (Tcf-1) expression is decreased not only in TFH cells, but also in Aiolos-deficient TH1 cells that also display an increased cytotoxic gene signature ( Figs. 2A, 4G). This is consistent with the findings from Donnarumma et al. Cell Reports 2016, demonstrating that TCF-1 expression can oppose the acquisition of cytotoxic features by CD4 T cells.

Tcf1 acts as a master transcriptional regulator of T cells, with expression in naïve T cells and maintenance in Tfh cells. Is Tcf1 reduced in naïve cells or only specifically in Tfh cells in Aiolos-deficient mice? Is downregulation of Tcf1 only observed under TFH-like polarizing culture conditions? It would be good to know whether this is specific for Tfh cells. Is the phenotype of Aiolos knockout mice like that of Tcf1 knockout mice, and can overexpression of Tcf1 or other targets rescue the Aiolos-deficient phenotype? Is regulation of Tcf1 by Aiolos universal or specific to Tfh cells? New transcript data from wildtype versus Aiolos-deficient naïve CD4 + T cells demonstrate that the effect of Aiolos deficiency on TCF-1 expression does not extend to naïve T cells (Extended
Minor Points (as indicated by Reviewer 1)

Il2ra is increased in Ikzf3-/-cells; however, the effect is relatively small (~1.5-fold), suggesting IL-2ra may not be a primary Aiolos target.
We appreciate the comment from the Reviewer and understand the concern over the small transcriptional effect. However, we now show that in the absence of Aiolos, CD25 and CD122 expression are augmented at the transcript level in vitro (Figs. 2B, 4G, Extended Data Fig. 13A), and at the protein level in vivo (Figs. 5F, 6H-I, Extended Data Figs. 9F, 10A). Conversely, we now show that overexpression of Aiolos represses expression of both Il2ra and Il2rb at the transcript level (Extended Data Fig. 13B). Functionally, we observe that Aiolos deficiency results in increased STAT5 tyrosine phosphorylation-mediated activation in both TFH and TH1-polarized cells (Fig. 8A, Extended Data Fig. 13C). Our expanded ATAC-seq data also support a role for Aiolos in regulating CD25 (and CD122) expression (Figs. 3A, 7A-C, and Extended Data Fig. 12).
Despite relatively modest transcript changes, these findings strongly support a phenotypic and functional effect for Aiolos-dependent alterations at the Il2ra and Il2rb loci. Figure 7)

The paper dissects the "Zfp831/Tcf-1/Bcl6 axis", yet this is not shown in the graphic model (Extended Data
We thank the Reviewer for this observation and have made necessary adjustments to the graphic model (Extended Data Fig. 14).

The authors show the Tfh associated gene expression in in vitro cells cultured under Tfh-like polarizing
conditions. How about the expression of Tcf-1, Bcl-6, and Zfp831 in primary Tfh cells from wild-type and Aiolos knockout mice?
We agree with the Reviewer that this is an important point. While we have not performed a comprehensive analysis on all TFH transcriptional regulators (analysis of Zfp831 is hindered by the lack of a commercially available flow cytometry antibody), our updated analysis of Bcl-6 protein expression during influenza virus infection (6 days post infection) reveals a statistically significant reduction in Bcl-6 protein in the absence of Aiolos (Extended Data Fig. 3B). These data are suggestive of an early TFH defect. We look forward to examining the expression of additional key TFH transcription factors at the protein level in future work.

The Stat5
ChIP-seq peak is missing in the IGV figure (Extended Data Figure 6). IGV figures have been substantially expanded, and each track (where applicable), includes STAT5 ChIP-seq tracks ( Fig. 7B-F, Extended Data Fig. 12).

REVIEWER 2 In this manuscript Read KA and colleagues investigated the role of the transcription factor Aiolos in Tfh cells. In a previous study from the same group (Read KA J Immunol 2017), the authors found that Aiolos expression was increased in antigen-specific Tfh cells after influenza infection, compared to antigen-specific effector T cells. This new study now aims at extending on this finding, by showing that in the absence of Aiolos, Tfh cell differentiation in response to influenza antigens was impaired, leading to reduced antibody response. On the other hand, T cells lacking Aiolos that were cultured under Tfh conditions acquired some features of a cytotoxic-like program, including perforin expression. Aiolos deficiency also resulted in increased CD25
expression.

While some observations warrant further investigation (for example, the role of Aiolos in CD4-CTLs, or in T cell responses to viral infections), other findings appear to be more incremental and preliminary, and in the absence of more thorough and global analyses I remain uncertain whether some of the conclusions are indeed adequately supported by experimental evidence.
We thank the Reviewer for their feedback and agree that more thorough and global analyses were required to support our findings. These include: 1. Global ATAC-seq analyses for WT vs. Aiolos-deficient TFH-and TH1-polarized cells (Extended Data Figs. 5, 11). 2. Analysis of statistically significant differences in accessibility throughout the genomes of WT vs. Aiolosdeficient cells (Figs. 3A, 7A).

Expanded analyses of full gene loci of interest (Extended
These have been incorporated into the substantially revised manuscript and are further detailed in the Authors' Summary and throughout this document. Also, please note that Point 1 has been subdivided based on each study that the Reviewer refers to.

Currently this study is not adequately put into the context of previous relevant work on Aiolos published by other labs. Most notably: -Quintana et al (Nature Immunology 2012) showed that Aiolos controlled the differentiation and function of Th17 cells, and that Aiolos was most highly expressed in this subset. In Figure 2a, how does expression of Aiolos in Tfh cells compare to Th17 cells? This information would provide more general insights on its role in T cell differentiation.
We now provide these data and find that Aiolos expression is elevated in both TFH and TH17 cells relative to other T helper cell populations (Extended Data Fig. 1A-B). Further, we have adjusted language in both the Results and Discussion sections to more thoroughly place our findings into the context of (or, rather, compare and contrast with) previous work in TH17 populations. It is also important to note that our analyses are performed in the context of infection, rather than autoimmune settings (as with prior TH17 cell work), which is an important distinction. Figure 1, can the authors rule out that reduced antibody production is not due to an intrinsic B cell defect? As discussed in response to Reviewer 1, while our in vitro data are strongly supportive of a CD4 cell-intrinsic role for Aiolos in regulating TFH programming, we cannot rule out some contribution of B cells or regulatory T cell populations to the observed reduction in TFH cell populations and antibody production. We also now include a discussion of the above work, though it is important to note that the AIOLOS mutation in question resulted in a dominant negative form of Aiolos, rather than its deficiency, as explored in the current manuscript.

-Kuehn et al (J Exp Med 2021) showed that a mutation in AIOLOS identified in human patients was associated to T and B cell abnormalities (including impaired Tfh cell differentiation), recapitulated in a mouse model. In
-Wang JH (Immunity 1998) showed increased proliferation of Aiolos-deficient T cells in response to TCR activation. Can the authors rule out that some of the differences they observe in the current study are not due differences in T cell activation?
We appreciated this feedback from the Reviewer and acknowledge that Aiolos has previously been implicated in regulation of T cell activation. To address whether this was also the case in our analyses, we analyzed expression of numerous cell activation markers by Aiolos-deficient TH1 and TFH-polarized cells (Extended Data Fig. 8C-D). Our findings do not indicate that a global change in cell activation is responsible for the observed phenotypes. Furthermore, we do not observe a significant difference in the frequency or number of antigenspecific populations in either the DLN or lungs following adoptive transfer of wildtype or Aiolos-deficient populations, suggesting again that global T cell activation/proliferation differences are not driving the observed differences in TFH and CTL populations ( Fig. 6G and Extended Data Fig. 10E).

Figure 3: since the authors performed ATAC-seq on Tfh cells, they should assess if a more comprehensive analysis (rather than just looking at a few pre-selected genes) provides more information about the regulatory roles of Aiolos in Tfh cells. In general, I don't think that based on a few selected snapshots the authors can
draw the conclusion that "chromatin accessibility was negatively impacted by the absence of Aiolos". There might be many more regions where chromatin accessibility is positively impacted. And the slight reduction shown could simply reflect experimental variability. Also, how do these differences compare to differences in accessibility at established Aiolos targets, like Il2?
We appreciate this feedback from the Reviewer and agree with this assessment. As noted above, we have now performed genome-wide ATAC-seq analyses, and indeed observe global changes in chromatin accessibility (both augmented and reduced) in the absence of Aiolos (Extended Data Figs. 5, 11). Importantly, these included statistically significant alterations in accessibility at key target loci (Figs. 3A, 7A). Full loci are also presented to strengthen our observations, and regions of significantly altered accessibility have been noted (Extended Data Figs. 6, 12). In contrast to key TFH and CD4-CTL associated genes, we do not observe differences in accessibility at the Il2 locus in the absence of Aiolos in this setting (Extended Data Figure 12). We speculate that this may be due to 1) differences between autoimmune (Quintana et al. Nat Immunol 2012) and infection (present study) settings, and/or 2) differences in IL-2 expression in TFH vs. TH17 cells, as TFH cells, unlike TH17 cells, are known IL-2 producers (DiToro et al. Science 2018). Figure 3, a better control of specificity for the ChIP-PCR assay is represented by the Ikzf3-/-Tfh cells, which should be used in comparison with the other experimental groups.

Still in
We agree with the Reviewer, and now present these data here. These findings (at Zfp831) demonstrate that limited background for Aiolos IP is detected in Aiolos-deficient cells.
ChIP analysis of Aiolos enrichment at an intronic Zfp831 enhancer element in WT vs. Aiolos-deficient samples. Data are compiled from 3 independent experiments; samples were normalized to total input and are presented as percent enrichment relative to total control (n=3 ± s.e.m).

The authors could establish if Aiolos indeed directly regulates the expression of some of the identified factors (Bcl6, Il2ra, etc) by using gain-of-function experiments and luciferase reporter assays.
We appreciate the suggestion from the Reviewer and have indeed shown that Aiolos augments the promoter activity of Bcl6 in a prior publication (Read et al., J Immunol 2017). Further, we now include Aiolos overexpression data in TH1 cells, which indicates that overexpression of Aiolos is sufficient to repress both Il2ra and Il2rb transcript expression (Extended Data Fig.13B, and shown below). Conversely, we observe that overexpression of Aiolos results in a trending increase in the expression of the TFH gene Zfp831 (shown below), which is downregulated in the absence of Aiolos (Figs. 2B, 4G), and which we have identified as a direct Aiolos target (Fig. 3D).

The authors state (page 10) that Aiolos directly regulates CD25 expression. However, the authors only showed that the Il2ra transcript is expressed comparatively at higher levels (~2 times higher) in Ikzf3-/-Tfh cells compared to wild-type. How does this translate into the dynamics of CD25 expression at different time points of T cell activation and with different strengths of stimulation? A small upregulation of CD25 expression may end up being modest and primarily linked to increased T cell activation, rather than differentiation. B cells lacking Aiolos for example exhibited an activated phenotype (Wang JH Immunity 1998).
We appreciate this feedback from the Reviewer. As discussed above, our data are not consistent with overall increases in CD4 + T cell activation (Extended Data Fig. 8C-D). As included in response to Reviewer 1 point 4, our combined in vitro and in vivo expression data (Fig. 2B, 4G, 5F, 6H-I, Extended Data Figs. 9F, 10F, 13A-B) when coupled with the observed changes in STAT5 tyrosine phosphorylation (Fig. 8A, Extended Data Fig.  13C), support a strong phenotypic and functional effect of Aiolos with regard to the repression of IL-2/STAT5 signaling. Furthermore, our ATAC-seq and ChIP data (Figs. 3A, 7A-C, Extended Data Fig. 12, 13D) support a direct role for Aiolos in regulating IL-2Ra expression.
6. Along the same line, while I appreciate that the authors show increased CD25 expression in vivo (Figure 7), this could also be due to lower thresholds of T cell activation in absence of Aiolos. What about the surface expression of other activation markers (both in vivo and in vitro) that are not regulated by Aiolos? Including also Icos, which is central to Tfh biology. Our data demonstrate that activation markers, including Icos, are not universally upregulated by Aiolosdeficient cells (Extended Data Fig. 8C-D), as detailed in response to Reviewer 2, point 1.

7.
Please also indicate where all the relevant transcripts (including Il2ra,Il2rb,Icos) are located in the volcano plots in Figure 2 and 5.
We agree that this information should be included; these labels have now been added to the volcano plots ( Fig. 2A, Extended Data Fig. 7D).

A more in-depth comparison of the RNA-seq data in Tfh and Th1 cells could provide more information about the role of Aiolos in these cells. What is the extent of overlap of the up-and down-regulated genes in Tfh and
Th1 conditions in the absence of Aiolos? Some Venn diagrams with indicated common and unique genes would already be informative. If mostly the same genes are dysregulated in these two cell subsets upon Aiolos deletion, then the main phenotype may be linked to general T cell activation rather than differentiation. We agree that expanded analyses of RNA-seq findings would be beneficial. We have now analyzed unique and shared genes between up-and down-regulated genes in TH1 and TFH data (Extended Data Fig. 8A-B). These findings are now included and indeed demonstrate that while there are some similarities in the genes regulated by Aiolos expression in TH1 and TFH cells, there are also many differences.  Figure 6F: was Cx3cr1 expression increased also in any of the RNA-seq datasets? Excel files containing at the very least the significant differentially expressed genes and differentially accessible regions should be made available to be able to assess the consistency and quality of the data. The number of biological replicates used in RNA-seq for Tfh cells is not indicated neither in the methods nor in the figure legends. Please show PCAs of all RNA-seq and ATAC-seq experiments. Legend of Figure 3 mentions two replicates for ATAC-seq, which is insufficient to achieve robust results. The requested data have been added as an additional supplementary file and now include:

9)
1. The top 200 significantly differentially expressed genes for each cell type 2. PCA plots for all ATAC-seq and RNA seq analyses 3. Expanded biological replicates for TH1 ATAC-seq samples (n=3), and associated analyses 4. The top 500 significantly differentially accessible regions for each cell type 5. The top 60 enriched motifs within regions of both increased and decreased accessibility for each cell type Additionally, while we found the Cx3cr1 data to be encouraging, we have instead chosen to focus on more established markers of CD4-CTLs including NKG2A/C/E (Workman et al. Plos One, 2014 andMarshall et al. J Immunol, 2017) in the revised manuscript (Figs. 4G, 6E, 7A and Extended Data Fig. 12).

10) Based on the CD25 data presented in this manuscript, and in the absence of other information (that is, expression of other activation markers not regulated by Aiolos, functional effects of IL-2 on cell proliferation
and so on), the conclusion that "Aiolos represses IL-2 responsiveness" (page 12) is both premature and preliminary, since no direct evidences are presented. We agreed with the Reviewer that expanded analyses would strengthen our findings regarding the role of Aiolos in regulating IL-2 responsiveness. As discussed above, we now include: 1. Global ATAC-seq analyses, which identify regions of both Il2ra and Il2rb as statistically significant in terms of differential (enhanced) accessibility in the absence of Aiolos (Figs. 3A, 7A-C, Extended Data Figs. 5, 11). 2. Assessment of numerous activation markers, which indicate that Aiolos deficiency does not globally impact T cell activation (Extended Data Fig. 8C-D). 3. Analysis of CD25 and CD122 surface expression during influenza virus infection (Figs. 5F, 6H-I, Extended Data Figs. 9F, 10A). 4. Analysis of STAT5 tyrosine phosphorylation-mediated activation in the absence of Aiolos (Fig. 8A,  Extended Data Fig. 13C). 5. Broad analysis of STAT5 enrichment at CD4-CTL gene targets (including Il2ra) in the absence of Aiolos ( Fig. 7B-F, Extended Data Fig. 12).
Importantly, we do not observe alterations in accessibility at the Il2 locus in the absence of Aiolos (Extended Data Fig. 12). Together, these data are strongly supportive of a role for Aiolos in regulating IL-2 responsiveness and downstream STAT5 activation/activities.

I am uncertain what conclusions can be drawn from Figure 8: is the increased STAT5 phosphorylation actually due to IL-2 stimulation? Is it significant? Does it occur also in Aiolos-deficient Tfh cells? Does it affect T cell responses to IL-2?
Our data in Fig. 8A and Extended Data Fig. 13C demonstrate that STAT5 phosphorylation is augmented in the absence of Aiolos. These data when combined with increased IL-2R expression and the pathway analysis in both Aiolos-deficient TH1 and TFH cells (Fig. 2C, 4I) support the assertion that IL-2 signaling (but not production, as shown in Figure 5G) is increased in the absence of Aiolos. Furthermore, we find enhanced STAT5 enrichment at CTL genes in the absence of Aiolos (Fig. 8B-D). Finally, using HOMER motif analysis, we find that the STAT5 DNA-binding motif is one of the most overrepresented motifs in areas of significantly increased chromatin accessibility in the absence of Aiolos (Figs. 3F, 7G). We have altered the language describing our findings to be as precise as possible.

12) If Aiolos indeed suppresses CD25 expression, then its retroviral transduction in wt and Aiolos-deficient cells should reduce (or normalize, respectively) CD25 expression and any downstream functional effects.
As discussed in point 4, we now include Aiolos overexpression data in TH1 cells, which indicates that overexpression of Aiolos is sufficient to repress both Il2ra and Il2rb transcript expression (Extended Data Fig.  13B).

13) The observation that T cells lacking Aiolos express granzyme and perforin is interesting: do they also acquire killing capacity in vitro?
In our revised manuscript, at 8 days post-influenza infection in the adoptive transfer model, we generate wholetissue homogenates containing donor CD45.2-OT-II WT or Aiolos-deficient cells, which are stimulated ex vivo with OVA peptide. We find that in response to antigen stimulation, Aiolos-deficient cells produce increased IFN-g, perforin, and granzyme b, relative to their WT counterparts, suggestive of increased killing capacity ( Fig.  6F and Extended Data Fig. 10B-D).
14) It is unclear from the figure legend if the human data shown in Extended Figure 1 show 3  We appreciate that the Reviewer found the data to be of good quality. We also appreciate the thorough critique and suggestions for improving the study. We have now obtained new data that both expand the scope of our study and strengthen our original conclusions. We appreciate the feedback from the Reviewer and now present data from the lungs (in an adoptive transfer setting) showing that cells with augmented cytotoxic features are indeed significantly increased in Aiolosdeficient transferred populations relative to their WT counterparts ( Fig. 6 and Extended Data Fig. 10B-D).
Further, data demonstrating reduced weight loss in Aiolos-deficient animals suggest that they may be resistant to influenza infection (presented below). Fig 1E). In light of the role of Aiolos inhibiting CD25, does Aiolos also control Treg formation?

The authors show increased Tregs (Ext
We appreciate this question from the Reviewer and find (as discussed in detail in response to Reviewer 1), that Foxp3 + TREG cells are significantly elevated in the absence of Aiolos (Extended Data Fig. 3D-E). We are currently exploring this relationship more thoroughly in a separate study. It is important to note that in our adoptive transfer model we see normal TREG numbers as well as a strong increase in CD4-CTL programs in the lungs (as detailed above).

How does Aiolos alter chromatin accessibility? Is there increased H3K4me3 or H3K27Ac? Given the use of an in vitro system and the authors ability to perform ChIP, this is addressable and would improve the impact with mechanism of how Aiolos alters gene transcription.
We appreciate these questions from the Reviewer and now find that our data are consistent with two mechanisms: 1. At key TFH gene loci (at sites of observed Aiolos enrichment in WT cells), loss of Aiolos correlates with a significant decrease in H3K27Ac ( Fig. 3E and Extended Data Fig. 6E). 2. In contrast, at genes associated with the CD4-CTL differentiation program, we observe not only increased STAT5 enrichment, but also increased H3K27Ac in the absence of Aiolos (Fig. 8E).
Collectively, these findings suggest that Aiolos positively impacts chromatin accessibility at TFH genes, and negatively impacts chromatin accessibility at CTL genes via suppression of IL-2 signaling and STAT5 enrichment.

NP specific tetramer staining should be shown by flow.
We apologize for this omission. These data are now included in our gating strategy (Extended Data Fig. 3A).

When is Aiolos upregulated? At what point in T cell differentiation is it required for expression? Tfh cells are thought to form early, and if Aiolos is upstream of Bcl6, it suggests Aiolos is required early.
New data demonstrate that Aiolos expression is upregulated 24h after cell activation and correlates positively with expression of the key TFH transcriptional regulator Bcl-6 (Extended Data Fig. 1E-F). Importantly, this upregulation of Aiolos was not observed in TH0 cells (cultured on stimulation, but under non-polarizing conditions), suggesting that Aiolos expression in induced by cytokine, not stimulation, signals.

If Aiolos is upregulated and not expressed in naïve cells, what drives its expression?
A prior study has shown that Aiolos expression was regulated by STAT3 and Ahr in TH17 cells (Quintana et al. Nat. Immunol. 2012). We are currently interrogating these possibilities in TFH cells (i.e. IL-6/STAT3) in a separate study. However, our findings in the current study (discussed in point 5) suggest that cytokine signals are required for the induction of Aiolos.

For all in vivo experiments, cell numbers should be shown as well as percentages.
As detailed in our response to Reviewer 1, we have now included the numbers of evaluated influenza nucleoprotein (NP)-specific (or CD45.2 + donor T cell, or CD45.2recipient) populations in each tissue examined (Figs. 1D, 5E, 6G and Extended Data Fig. 3C, 9C, and 10E-G).